Escherichia coli: The Trp Operon
Tryptophan is produced by the enzyme tryptophan synthase, which is encoded by the tryptophan operon. The Trp operon consists of a group of genes, including an alpha and beta subunit (trpA and trpB), as well as trpC, D, and E [3]. The Trp operon was the first repressor operon to be discovered, and it is repressed by the presence of tryptophan [2]. The trpR gene (also part of the operon) produces a regulatory protein responsible for repression. Transcription normally starts at the operator region of the operon. This region can also be repressed, thereby blocking transcription [2]. A schematic of the Trp operon layout is depicted in Figure 1. Trp operon transcription is dictated by an interaction between operator and repressor components. Studies by Yanotsky and colleagues have demonstrated that the RNA polymerase and repressor are found to have interactions in the same area of the Trp operon [1 ]. Trp promoter-operator The promoter-operator portion of the Trp operon was identified through transcription by a trp tra nsducing phage and subsequent sequence analysis [1 ]. This was executed using strain W3110 trpE9829. A phage was used to infect the bacteria. 32PO4 was present in order to label the RNA. 3 hybridizations were performed in order to successfully isolate the complemenatary RNA piece to the region bearing the trp promoter-operator [1 ]. Gel electrophoresis was performed, with subsequent audioradiography to identify the RNA bands. RNA fingerprinting was utilized in order to separate products from RNAase T1 and RNase A digestions. RNase T1 is an endoribonuclease that cleaves single-stranded RNA at guanines. RNase A cleaves single-stranded RNA at C and U residues. These endoribonucleases were used to identify the oligonucleotide sequence of the trp promoter-operator region. An example of RNA fingerprinting for the trp promoter-operator region is depicted in Figure 2. Trp operon repression An article from Zubay and colleagues in 1972 helped to elucidate the function of the trpR gene and how it plays a role in repression. To study the effect of the Trp repressor, beta-galactosidase production was used as a readout for activity. A bacteriophage was used to transduce the beta-galactosidase gene of the operon, but with trp control. A fusion operon was created by joining the trp operon and the lac operon of E. coli. The fusion operon includes the operator, a portion of the trpE gene, followed by lac Z, y, and a genes [2]. Obtaining the Trp repressor To study the activity of the TrpR gene, isogenic strains of Escherichia coli were created which only differed by having the trpR+ allele (the active repressor) in one and the trpR- allele in the other. A cell free extract was generated through a S-30 method, in which cells were ground up and then centrifuged at 30G in order to retrieve the solubl e components of the cell. This method generated extracts with either the presence or absence of trpR. Creation of beta-galactosidase-containing strains A lamda-phage which produces beta-galactosidase under the control of the Trp regulatory system was used to create strains that produce beta-galactosidase when the Trp regulatory system is activated, but not when it is repressed. This construct is referred to as trp-lac fusion DNA. When S-30 trp+ extracts described in the section above were applied at increasing concentrations to stains containing the trp-lac fusion DNA, beta-galactosidase activity decreased. This result is highlighted in Figure 3. References [1 Bennett G., Schweingruber M., Brown K., Squires C., and Yanofsky C. Nucleotide sequence of the promoter-operator region of the tryptophan operon of Escherichia coli. J Mol Biol 1978 (121) 113-137.] [2]Zubay G., Morse D., Schrenk W., and Miller J. Detection and isolation of the repressor protein for the tryptophan operon of Escherichia coli. Proc Nat Acad Sci 1972 (69) 1100-1003. Trp Operon Wiki